Download Extended Survivability of Prostate Cancer Cells

(CANCER RESEARCH 58. 3466-3479.
August 1. 1998]
Extended Survivability of Prostate Cancer Cells in the Absence of Trophic Factors:
Increased Proliferation, Evasion of Apoptosis, and the Role of Apoptosis Proteins
Dean G. Tang,1 Li Li, Dharam P. Chopra, and Arthur T. Porter
Department of Radiation Oncology, Wayne State University, Detroit, Michigan 48202 ID. G. T., A. T. P.¡; Karmanos Cancer Institute, Detroit, Michigan 48201 [D. G. T.,
A. T. P.]; Biomide Corporation, Grosse Pointe Farms. Michigan 48236 [D. G. T., L. L.]; and institute of Chemical Toxicologv, Wavne State University, Detroit, Michigan 48226
ID. P. C.I
ABSTRACT
onstrated an overall survivability in the order of BPH < NHP < LNCaP
< PC3 < PCA < Du 145. Upon growth factor deprivation, NHP/BPH cells
rapidly underwent apoptosis, leading to a decreased number of live cells.
PCA/PC3/Dul45
cells, in contrast, demonstrated an initial phase of ag
gressive growth during which apoptosis rarely occurred, followed by a
"plateau" phase in which cell loss by apoptosis was compensated by cell
accepted that apoptosis is a dominant feature unless suppressed or
overridden by growth/survival factors; therefore, most eukaryotic
cells (with the exception of, probably, blastomeres) will undergo
apoptosis when deprived of appropriate survival factors (1, 2). In
creased cell proliferation, extended cell survival, and diminished
apoptosis have all been implicated in tumorigenesis.
There has been a close association between apoptosis and prostate
cancer initiation, progression, metastasis, and response to treatment.
The growth and survival of normal prostate epithelial cells depend on
androgens; therefore, castration or chemical blocking of androgen
function leads to prostate atrophy. Prostate cancer is composed of
androgen-dependent and androgen-independent cells. Androgen abla
tion eliminates most androgen-dependent cancer cells by inducing
proliferation, followed by a later phase in which apoptosis exceeded the
cell proliferation. LNCaP cells demonstrated survival characteristics be
tween those of NHP/BPH and PCA/PC3/Dul4S cells. We concluded that
the increased survivability in prostate cancer cells results from enhanced
cell proliferation as well as decreased apoptosis. The molecular mecha
nisms for evasion of apoptosis in prostate cancer cells were subsequently
investigated. Quantitative Western blotting was used to examine the pro
tein expression of PS3 and P21WAIM, Bcl-2 and Bcl-XL (anti-apoptotic
apoptosis but can rarely cure the patients due to the presence of
androgen-independent
cells and emergence of apoptosis-resistant
clones (3, 4). Bcl-2 oncoprotein expression has been correlated with
the progression and metastasis of prostate cancer cells and the emer
gence of androgen-independent cells, as well as the generation of
apoptosis- and therapy-resistant cancer cell clones (5-11). On the
other hand, androgen-independent prostate cancer cells still retain
proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results
revealed that, upon trophic factor withdrawal, NHP and BPH cells upregulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and
down-regulated the expression of P21. Furthermore, NHP and BPH cells
endogenously expressed little or no Bcl-2. In sharp contrast, prostate
cancer cells expressed nonfunctional P53 and various amounts of Bcl-2
proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2
and/or BciXL, lost response to withdrawal-induced up-regulation of Bax/
appropriate apoptotic machinery and can be induced to undergo
apoptosis by a wide spectrum of biological and pharmacological
treatments, including wild-type P53 and P212 (12-14), prostate
This project was undertaken to study the survival properties of various
prostate cells, including normal (NHP), BPH (benign prostate hyperplasia), primary carcinoma (PCA), and metastatic prostate cancer cells
(LNCaP, PC3, and Dul45), in the absence of trophic factors. Cell prolif
eration and cell death were quantitated by enumerating the number of live
cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-diamidino-2-phenylindole
dihydrochloride
nuclear staining. These cells dem
Bad/Bak or decreased or even completely lost Bax expression and ex
pressed some novel proteins such as P25 and P54/55 complex. These data
together suggest that prostate cancer cells may use multiple molecular
mechanisms to evade apoptosis, which, together with increased prolifer
ation, contribute to extended survivability of prostate cancer cells in the
absence trophic factors.
INTRODUCTION
Tissue and organ homeostasis is controlled by balanced cell pro
liferation, cell survival, and cell death. Cell proliferation is driven and
regulated by various peptide growth factors and/or cytokines. Cell
survival represents the cell's intrinsic life expectancy in the absence of
survival factors, many of which overlap with the growth factors/
cytokines that drive cell proliferation. Physiological cell death occurs
mostly via programmed cell death or apoptosis (1). Apoptosis is a
genetically controlled suicidal program that takes place, in many
cases, when the cells run out of their survivability in the absence of
growth/survival (or trophic) factors, although it can also be triggered
by a wide variety of physiological or obnoxious stimuli. It is generally
Received 2/5/98; accepted 6/2/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Department of Radiation
Oncology, Wayne State University, 407 Life Science Building, Detroit, MI 48202. Phone:
(313)577-2184; Fax: (313)577-2375; E-mail: [email protected].
apoptosis response-^ (15), prevention of adhesion (16), transforming
growth factor ß(17), tumor necrosis factor a (18), Fas (19), chemotherapeutic drugs such as cisplatin, camptothecin, teniposide, and
vincristine (20, 21), novel chemical entities such as Linomide (quinoline-3-carboxamide; Ref. 22), ß-lapachone (23, 24), phenylbutyrate
(25), ribonucleotidase inhibitors (26), diethylstilbestrol (27), ß-L-(-)dioxolane-cytidine (28), and retinoid derivatives (29), and signal
transduction modifiers such as phorbol esters (30-32) and thapsigargin (33). We recently reported apoptosis induction by prostate secre
tory protein 94 (34) and hydroxamic acid compounds (35, 36) in
hormone-refractory human prostate cancer cells.
Little information exists in regard to differential properties of
normal and cancerous prostate epithelial cells in terms of sensitivity to
apoptosis induction. Furthermore, no systematic studies have been
performed to investigate the interrelationship among the proliferation,
survival, and apoptosis of various types of prostate cells. Recently, a
series of normal (NHP2), BPH, and PCA prostate cultures have been
established and characterized (37). In this study, we took advantage of
these primary cultures and compared their growth and survival prop
erties in the absence of trophic factors with those of the established
malignant prostate cancer cell lines, i.e., LNCaP, PC3, and Dul45.
We further investigated the changes and the potential roles of several
apoptosis proteins in these six different cell types upon growth factor
withdrawal. The results show that prostate carcinoma cells demon
strate a much higher proliferative potential and survive much longer
than normal and BPH cells.
2 The abbreviations used are: NHP, normal human prostate; BPH, benign prostate
hyperplasia; PCA, primary prostatic carcinoma; DAPI, 4',6-diamidino-2-phenylindole;
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
bromide; P21, P21WAIM.
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APOPTOSIS
MATERIALS
AND PROSTATE
form formazan dyes. Viable cells with active mitochondria cleave the tetrazo-
AND METHODS
Cell Culture, Antibodies, and Reagents.
CANCER CELL SURVIVAL
PC3. Du 145, and LNCaP (FGC-
10) cells were obtained from American Type Culture Collection and routinely
cultured in RPMI 1640 supplemented with 10% fetal bovine serum. 2 mM
L-glutamine. and antibiotics. NHP, BPH. and PCA cells were surgically
isolated from clinically diagnosed and pathologically confirmed patients and
characterized as detailed previously (37). All of these primary cells were
cultured in KBM basic medium (Clonetics) supplemented with 5 fig/ml
insulin. 0.5 /^g/ml hydrocortisone. 10 ng/ml epidermal growth factor, 50 /J.g/ml
bovine pituitary extract. 10 ng/ml cholera toxin, and antibiotics (37). Only
cultures of passages 2-5 were used throughout this study. The following
primary antibodies were used in the immunoblotting experiments, (ai anti-p53:
a mouse monoclonal anti-human wild-type p53 (clone DO-1; amino acids
21-25 as the immunogen) from Oncogene Research/Calbiochem and another
mouse monoclonal anti-human wild-type p53 (clone DO-1 ; amino acids 37-45
as the immunogen) from Santa Cruz Biotechnology: (b) anti-p21: a rabbit
polyclonal anti-human peptide antibody (amino acids 146-164 as the immuno
gen) from Santa Cruz: (c) anti-Bcl-2: a mouse monoclonal anti-human peptide
Bcl-2 (amino acids 41-54 as the immunogen) from either Dako (clone 124) or
Calbiochem (clone 100), and a rabbit polyclonal anti-human Bcl-2 raised
against a peptide corresponding to amino acids 4-21 (sc-492) from Santa
Cruz; (d) anti-Bcl-Xs/I : a rabbit polyclonal anti-human peptide antibody
(amino acids 2-19 as the immunogen) from Santa Cruz; (e) anti-Bax: a rabbit
polyclonal anti-human Bax peptide antibody (amino acids 150-165 as the
immunogen) from Calbiochem and a rabbit anti-human Bax peptide antibody
(amino acids 43-61 as the immunogen) from PharMingen; (/) anti-Bak: a
rabbit polyclonal anti-human Bak (amino acids 82-104 as the immunogen)
from Santa Cruz; (#) anti-Bad: a rabbit polyclonal anti-mouse Bad (amino
acids 185-204 as the immunogen) from Santa Cruz; and (/i) anti-actin: a mouse
monoclonal anti-actin from ICN. The secondary antibody was either goat
anti-rabbit or anti-mouse IgG (whole molecule) conjugated to horseradish
peroxidase (Amersham). ECL reagents were bought from Amcrsham. The
Western blotting experiment was performed as described previously (38. 39).
For starvation, various cells cultured in regular media were washed (three
times) with growth factor-free RPMI medium followed by culturing cells for
different periods of time in RPMI.
Quantification of Live Cells Using the MIS Assays. Cell survival was
determined using the CellTiter MTS/PMS cell proliferation kit (Promega
Corp.), as described (39). Briefly, log-phase growing prostate cells were
cultured in Hat-bottomed 96-well plates (1 X IO4 cell/well) in either RPMI-
lium ring into a visible dark blue formazan reaction product while dead cells
are not stained. The viable cells in each well were counted by bright-field light
microscopy.
Western Blotting. Western blotting protocols were detailed before (38.
39). Whole-cell lysates were prepared by directly scraping control or starved
prostate cells into the TNC lysis buffer [10 mM Tris-acetate (pH 8.0), 0.5%
NP40. and 5 mM CaCU], and solubilized proteins were analyzed on 4-20%
gradient or 12% SDS-PAGE. Isolated proteins were transferred to nitrocellu
lose and probed with individual primary antibodies as specified in "Results."
The secondary antibody used was goat anti-rabbit or -mouse IgG conjugated to
horseradish peroxidase. Then membranes were stripped and reprobed with
different antibodies. In some cases, the membrane was incubated with two
different primary antibodies. The intensities of individual bands were quantitated by densitometric scanning (41). and the values were normalized against
the intensity of actin.
Characterization of Bcl-2-related Proteins. The Bcl-2 protein normally
runs at M, —¿26.000
on SDS-PAGE under reducing conditions. Two prominent
upper bands (M, -27.000 and M, 30.000. respectively) were also detected in
this study using a monoclonal anti-Bcl-2 (raised against the 41-54 peptide of
human Bcl-2) in both PC3 and/or Dul45 cells (see "Results"). These upper
bands may represent phosphorylated
matched control monoclonal antibody MOPC-21 (IgGl) were added to the
precleared supernatants and incubated at 4°Cfor 2 h under rotating conditions.
At the end. 100 ¿dof protein A/G-agarose beads were added, and the mixture
was further incubated for l h at 4°C.The beads with bound antigen-antibody
complex were washed extensively: TNC lysis buffer (two times). TNC-0.1%
SDS (one time). TNC-1 M NaCI (one time), and TNC lysis buffer (one time).
Finally, the washed beads were reconstituted in 50 ¡¿I
of 2X SDS-PAGE
sample buffer [125 mM Tris-HCI (pH 6.8), 10% ß-mercaptoethanol. 4% SDS.
20% glycerol. and 0.001% bromphenol blue]. After boiling. 25 /il of the
precipitated sample were loaded onto a 12% SDS-PAGE. and separated
proteins were transferred to nitrocellulose membrane and then blotted using the
monoclonal or the rabbit polyclonal anti-Bcl-2. In the second set of experi
10% PCS medium (for LNCaP. PC3. and Dul45 cells) or in KBM medium
supplemented with various growth factors (for NHP. BPH. and PCA cells).
The wells were gently washed with plain media (times three), and then 200 /il
of trophic factor-free RPMI were added to each well to start the deprivation
ments. 50 /j.g of clarified PC3 or Du 145 cell lysates were incubated with 2
units of alkaline phosphatase at 37°Cfor 4 h. At the end. 25 /ig of the protein
were loaded onto a 12% SDS-PAGE. and separated proteins were used for
immunoblotting with the monoclonal anti-Bcl2. as described above.
experiment. The number of live cells on each day thereafter was determined by
the uptake of MTS/PMS dyes and measured on a 3550-UV microplate reader
(Bio-Rad) using the/1.,,,,, absorbance. One set of wells were measured just prior
to the media change, and the absorbance readings were used as the baseline
value (i.e., day 0). The cell number on each day was expressed as the
percentage cell number of the day 0. For each day's measurement, five- or
six-well repeats were run, and each experiment was performed two to six times
(see figure legends). A >100% cell number indicates cell growth during that
period.
Apoptosis Assays by Light Microscopy and DNA Fragmentation. These
methods have been detailed previously (35. 36. 38. 39).
Quantitation of Cell Death with DAPI Staining and MTT Assays.
Different types of prostate cells cultured on glass coverslips in the absence of
growth factors for various days were directly stained with DAPI (5 jag/ml) for
10 min at 37°C.At the end, coverslips were gently rinsed (two times) and
mounted onto slides with a small amount of SlowFade anti-fade mounting
media (Molecular Probes, Eugene, OR). The slides were observed under a
Zeiss fluorescence microscope and pictures were recorded on Tmax-400
black/white films. The cells showing condensed chromatin and/or fragmented
nuclei were scored as apoptotic. An average of 500-1000 cells were counted
for each slide/day. The results were expressed as the percentage of apoptosis
of day 0.
Alternatively, cell growth/death were monitored by MTT (40) assays. In this
assay, the MTT dyes are metabolized by mitochondria! dehydrogenases to
isoforms of Bcl-2, some novel Bcl-2-
related proteins, or simply nonspecific products. These possibilities were tested
with the following two sets of experiments. In the first. PC3 and Du 145 cells
were harvested, and cell lysates were prepared using the TNC lysis buffer, as
described above. Five hundred /ig of PC3 or Du 145 cell lysates were used for
immunoprecipitation
experiments, as described previously (41) with some
modifications. Briefly, the cell lysates in 1 ml of TNC lysis were first
precleared by incubating with 75 fil protein A/G-agarose (Santa Cruz Biotech
nology) at 4°Cfor 1 h. Then 5 /ig of the monoclonal anti-Bcl-2 or isotype-
RESULTS
Expression of Apoptosis Proteins in Various Prostate Cells. We
first examined the expression of various apoptosis-related proteins in
different types of prostate cells. The results are plotted in Fig. 1. All
cells except PC3 expressed P53, as revealed by a monoclonal anti
body. Note that longer exposure of the film also resulted in the p53
band in BPH cells (see also Fig. 7). P53 expressed in the primary cells.
i.e., NHP, BPH. and PCA, was shown previously to be wild-type (37).
LNCaP cells also express wild-type P53 (12). PC3 cells are P53-null;
therefore, no protein could be detected (Fig. 1). Dul45 cells expressed
the highest amount of P53 protein because the p53 gene in these cells
has two mutations (codons 223 and 274), resulting in accumulation of
the mutant protein (12). P21, a P53 target, was expressed in all of the
cells including PC3 (Fig. 1). There appear to be some differences in
terms of the protein amount of p2lWAF-' in different cells (Fig. 1).
When using 25-100 jug of whole-cell lysates, the M, 26,000 Bcl-2
protein was undetectable or. in some cases (e.g.. Fig. 3S), only faintly
detectable in NHP cells (Fig. 1). In similar experiments, Bcl-2 was not
detected at all in BPH and PCA cells (Fig. 1; see also Figs. 7 and 9)
3467
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APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
antibody (data not shown). Western blotting was also performed with
three proapoptotic proteins, Bax, Bad, and Bak. All of the prostate
cells expressed the M, -22,000 Bad (Figs. 3-7) and Mr -25,000 Bak
(Fig. 1). All prostate cells except Dul45 expressed the Mr -21,000
P53
P21
—¿
WAF-l
•¿21
kd
,30kd-Bcl-2
3cl-2 (27 kd)
x26-kd Bcl-2
Bcl-2
Bel-XL
—¿
Bax
Bak
.
products. We performed two sets of experiments to differentiate
among these possibilities. In the first, Bcl-2 and/or Bcl-2-related
proteins were immunoprecipitated, and the precipitates were further
subject to immunoblotting with the polyclonal or monoclonal antiBcl-2. This type of "double-shot" (i.e., immunoprecipitation followed
•¿30kd
~
^.
by immunoblotting) protocol has been widely used to confirm the
specificity of protein bands identified on Western blot. As shown in
Fig. 24, the MT 30,000 band was specifically immunoprecipitated
down by the monoclonal anti-Bcl-2 but not by control antibody
MOPC-21, suggesting that this protein represents a Bcl-2-related
21 kd
•¿25kd
Actin
Fig. 1. Expression of apoptosis proteins in prostate cells. Twenty five /ig of whole-cell
lysates prepared from regularly cultured primary [i.e., NHP (normal), BPH. and PCA
(primary) cells] or established prostate cancer cells (i.e., LNCaP, PC3. and Dul45) were
resolved on 4-20% SDS-PAGE and used for immunoblotting with a monoclonal anti-P53
(amino acids 21-25 as the immunogen). The membrane was sequentially stripped [62.5
IHMTris-HCI (pH 6.5), 2% SDS, and 100 mM 2-ME, l h at 50°C]and reprobed with:
rabbit polyclonal anti-p21v
, monoclonal anti-Bcl-2 (amino acids 41-54 as the im
munogen), polyclonal anti-Bcl-X^s, polyclonal anti-Bax (amino acids 43-61 as the
immunogen), polyclonal anti-Bak, polyclonal anti-Bad (not shown), or monoclonal antiactin (see "Materials and Methods" for details). The same panels of antibodies were used
in all of the subsequent experiments unless specified otherwise. As indicated in "Materials
and Methods" and other parts of the text, several different antibodies were sometimes used
for certain molecules for the purpose of characterization. The secondary antibodies used
were either goal anti-rabbit or anti-mouse IgG conjugated to HRP. Left, identities of
individual proteins; right, corresponding molecular weights. Actin is detected as a Afr
—¿45.000
protein. The monoclonal anti-Bcl-2 consistently detected several different bands
in LNCaP (M, 26,000 Bcl-2), PC3 (M, 26,000, M, 27,000, and M, 30,000 Bcl-2) and
Du 145 cells (M, 30,000 Bcl-2). The M, 27,000 band is labeled as the phosphorylated Bcl-2
(see Fig. 2).
in several repeat experiments, even after extended exposure. The M,
26,000 Bcl-2 protein was detected in LNCaP cells (Fig. 1). In PC3
cells, in addition to the Mr 26,000 Bcl-2, a Mr 27,000 and a Mr 30,000
protein were also detected (Fig. 1). In Dul45 cells, by contrast, only
the Mr 30,000 protein was detected (Fig. 1) in addition to the Mr
26,000 Bcl-2, which was detected only when higher amounts of
proteins/longer exposure were used (Fig. 2). This pattern of expres
sion for Bcl-2 protein was also observed when the membrane was
probed with a polyclonal antibody (data not shown). In general, the
Bcl-XL protein was detected as a faint Mr —¿30,000protein in all
prostate cells (Fig. 1, indicated by an arrow). This polyclonal antibody
also recognized two prominent upper bands, the identities of which
were unknown and whose intensities varied with different cell types.
No Bcl-Xs protein was detected in any cell type with this polyclonal
Bax protein, as revealed by an antibody recognizing the amino acids
43-61 of human Bax. The lack of Bax protein expression in Du 145
cells was also confirmed by another polyclonal anti-Bax raised against
peptide 150-165 (data not shown). Interestingly, LNCaP and PC3
cells appear to express more Bax proteins than the three primary
prostate cells (Fig. 1).
The monoclonal anti-Bcl-2 used in our studies did not recognize
any other Bcl-2 family proteins (Fig. 1 and data not shown). There
fore, the A/r 27,000 and MT30,000 bands detected by this antibody in
PC3 and/or Du 145 cells may represent phosphorylated Bcl-2 protein
(42-46) or a new Bcl-2-related protein, such as BRAG-1, which
migrates at M, —¿30,000
(47), or simply represent nonspecific reaction
protein instead of a nonspecific product. Blotting with the polyclonal
anti-Bcl-2 antibody produced similar results (data not shown). Note
that the status of the Mr 26,000 and Mr 27,000 proteins in this
experiment was not very clear due to the proximity of these bands to
the immunoglobulin light chain (MT—¿25,000;
Fig. 2A). The direct use
of whole-cell lysates in immunoblotting again identified the Mr
26,000, Mr 27,000, and M, 30,000 (on the order of Mr 26,000 » Mr
27,000 > Mr 30,000 in terms of intensity) protein bands in PC3 cells,
and Air 26,000 and Mr 30,000 (Mr 30,000 » Mr 26,000) protein
bands in Du 145 cells (Fig. 24). Only the Mr 26,000 Bcl-2 protein was
detected in A431 human epidermoid carcinoma cells (used as a
control; Fig. 24, Lane 1). In the second set of experiments, whole-cell
lysates from PC3 and Dul45 cells were treated with alkaline phosphatase and then immunoblotted with monoclonal anti-Bcl-2. As
shown in Fig. 2B, in PC3 cells, phosphatase treatment dramatically
decreased the amount of the Mr 27,000 protein with a simultaneous
increase in the quantity of MT 26,000 protein, suggesting that the MT
27,000 protein in PC3 cells is phosphorylated Bcl-2. In contrast, the
level of the Mr 30,000 protein in PC3 cells was hardly affected by the
phosphatase treatment, which similarly did not affect the mobility of
the Mr 30,000 protein in Dul45 cells (Fig. 20). The intensity of the MT
30,000 protein in Du 145 cells appeared to decrease a small amount;
however, no increased Mr 26,000 Bcl-2 protein was observed after the
phosphatase treatment (Fig. 2B). Taken together, these data suggest
that the Mr 27,000 protein represents the phosphorylated isoform of
Bcl-2 protein, and the Mr 30,000 protein may represent a novel
Bcl-2-related protein, the true molecular identity of which awaits
further characterization.
Survival of NHP Cells in the Absence of Trophic Factors. As
early as 24 h after the withdrawal of growth factors, NHP cells
stopped proliferation, as measured by MTS assays (Fig. 3A). There
after, interestingly, there appeared to have a slight "bouncing-back" in
the cell growth 48-72 h after culturing in the absence of trophic
factors (Fig. 3A), as observed consistently in several repeat experi
ments. The reason for this observation was not clear. DNA fragmen
tation assays (Fig. 3B, inset), DAPI staining (Fig. 3Q, and MTT
assays (not shown) revealed that NHP cells died through apoptosis.
3468
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APOPTOSIS
Anti-Bcl-2
Whole
cell lysates
s
97.4 —¿
66 —¿
46 —¿
30 —¿
21.5 —¿
AntiBcl-2
Blotting
MOPC
--pn
kî
à SriÃ
D
AND PROSTATE
CANCER CELL SURVIVAL
B
Anu-Bcl-2
I.P
PC3
pi
u
fi
+
U
—¿Ij; heavy
chain
^-*
•¿Â»â€¢*-i
light
97.466-
Cells
-
Alk. phos.
+
•¿â€” —¿Â» -BSA
4630-
chain
14.3—¿
-
Blotting
Dul45
21.5—¿
,-30kd-Bcl-2
=^©-Bcl-2 (27 kd)
Bcl-2 (26 kd)
Fig. 2. Characterization of Bcl-2-related proteins. In A. the M, 30,000 protein represents a specific Bcl-2-related protein. Five hundred /ig whole-cell lysates from either PC3 (Lanes
4 and 6) or Dul45 (¡Mnes5 and 7) cells were immunoprecipitated with either monoclonal anti-Bcl-2 (Lanes 4 and 5) or isotype-matched control antibody MOPC-21 (Lanes 6 and 7).
The immunoprecipitates were resolved on a 12% SDS-PAOE. and the membrane was probed with the monoclonal anli-Bcl-2. As controls, 5 fig of total cell lysates from A431 (human
epidermoid carcinoma; Lane /), 50 ng of whole-cell lysates from Du 145 (Lane 3). or 70 fig of lysates from PC3 cells (Lane 2) were directly used for Western blotting. Left, molecular
weight markers. Right. Ihe immunoglobulin heavy (M, ~54,000) and light (M, —¿25.000)
chains under reducing conditions. The M, —¿66,000
band in the whole-cell lysates (Lanes 1-3)
represents the BSA (the monoclonal anti-Bcl-2 was raised against a peptide conjugated to BSA). The Afr 30,000 Bcl-2 protein was precipitated by anti-Bcl-2 but not by MOPC-21
(arrowhead on the right). B, the M, 27,000 band is the phosphorylated Bcl-2 protein. For details, see "Materials and Methods" and other parts of the text. In untreated PC3 cells
(indicated by the "-" sign), the M, 26,000, M, 27,000, and M, 30,000 proteins were identified by the monoclonal anli-Bcl-2, which detected the M, 30.000 and the faint M, 26,000
protein in untreated Du 145 cells. Phosphatase (Alk. phos. ) treatment (indicated by the " + " sign) resulted in the disappearance of the M, 27.000 with simultaneous increase in the amount
of M, 26,000 Bcl-2 in PC3 cells, without affecting the mobility of the M, 30.000 band in either PC3 or Dul45 cells.
Increased apoptosis (i.e., positive DAPI labeling) was observed 24 h
after growth factor withdrawal. On day 2, —¿20%
cells were apoptotic
(Fig. 3, B and C, middle panel). By day 7, 60-70% cells were dead
(Fig. 3, B and C. right panel). Characterization of cell growth and cell
death by several different methods (i.e., MTS assays, DAPI staining,
DNA fragmentation assays, and MTT) overall produced consistent
results; NHP cells do not proliferate and undergo steady apoptotic
death upon removal of trophic factors (Fig. 3 and data not shown).
Note that the DNA ladder formation in all three primary cells (Fig. 3;
see also Figs. 6 and 8) was not as clear-cut as in established cell lines
(see Figs. 10, 12, and 14). This is not surprising because apoptosis in
primary cultures often is not accompanied by distinct DNA ladder
formation (reviewed in Ref. 4). Western blotting was used to examine
the changes of apoptosis proteins. As shown in Fig. 4, all proteins
except P21 increased immediately after deprivation, as determined by
densitometric scanning after normalizing the protein levels to that of
actin (Fig. 5: see legend for details). The most significant increase was
observed with Bcl-XL (> 15 fold by day 6; Figs. 4 and SA). The NHP
cells expressed barely detectable levels of Bcl-2, which appeared to
increase marginally by days 3 and 4 (Fig. 4). In contrast, all three
proapoptotic proteins, i.e., Bax, Bad, and Bak, significantly increased
with time (Figs. 4 and 5A). P53 also demonstrated a 2-3-fold increase,
whereas its target. P21, did not show any corresponding changes and
whose levels actually declined sharply by day 5 (Figs. 4 and 5/4).
Therefore, the P21 expression in NHP cells appears to be p53 inde
pendent.
Survival of BPH Cells in the Absence of Trophic Factors. Like
NHP cells, BPH cells did not grow, upon growth factor withdrawal, as
revealed by MTS assays (Fig. 6). In fact, BPH cells appeared to be
more sensitive than NHP cells to deprivation because they demon
strated an even faster loss in the number of live cells (compare Figs.
6A and 3/4). For example, —¿40%
of BPH cells were apoptotic (Fig. 6,
B and C, middle) by day 2 compared with —¿20%
apoptosis in NHP
3, after which its levels gradually decreased (Figs. 7 and 5ß).The P21
level did not undergo appreciable changes and was not affected by
P53 up-regulation. No Bcl-2 was detected in these cells (Fig. 7). The
Bcl-XL level was also very low, which showed only a marginal
increase (Figs. 7 and 5B). As in NHP cells, Bax and Bad proteins
persistently increased with time; however, Bak did not show any
changes (Figs. 7 and 5B).
Survival of PCA Cells in the Absence of Trophic Factors. PCA
cells behaved completely differently with respect to their survival in
the absence of trophic factors. Immediately (i.e., 24 h) after growth
factor withdrawal, PCA cells proliferated (>2.5 fold increase in cell
number) as determined by MTS assays (Fig. 8). PCA cells did not
demonstrate significant (15-20%) apoptosis until days 5-6, as sup
ported by faintly increased DNA ladder formation (Fig. 8ß,inset).
MTT assays (data not shown) also confirmed increased cell prolifer
ation and rare cell death. The total number of live cells did not
decrease, even by day 5 when increased apoptosis was observed (Fig.
8, B and C, middle panel), suggesting that continued cell proliferation
compensated for the cell loss. By day 11, nearly the same number of
cells (i.e., the same number of cells as initially input or 10,000
cells/well; see "Materials and Methods") were still alive (Fig. 8, A and
C, right panel). The most significant changes in apoptosis proteins in
PCA cells after trophic factor deprivation were the up-regulation of
p2]WAF-i and BCI_XL around day 4 (Figs. 9 and 5Q, prior to the
initiation of apparent DNA fragmentation (Fig. 8S, inset). Like NHP
and BPH cells, PCA cells expressed extremely low levels of Bcl-2,
which was undetectable (Fig. 9). In sharp contrast to NHP and BPH
cells, all other proteins, including P53, Bax, Bad, and Bak, were not
up-regulated (Fig. 9). However, interestingly, three prominent protein
bands, i.e., a MT -25,000 protein and a M, 54,000/55,000 doublet,
were simultaneously detected on day 4 after growth factor withdrawal,
when using the monoclonal anti-P53 for the Western blotting (Fig. 9,
top panel). These two novel entities of proteins were designated P25
cells at the same time (Fig. 3). By day 7, >80% cells were dead (Fig.
and P54/55, respectively.
Survival of LNCaP Cells in the Absence of Trophic Factors.
6, B and C, right panel). Western blotting revealed quite different
patterns of changes in apoptosis proteins than found in NHP cells. The
LNCaP cells demonstrated marginal proliferation upon serum starva
P53 up-regulation was most dramatic, with —¿30-fold
increase by day
tion, as revealed by MTS assays (Fig. 10/4). However, the cells did not
3469
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APOPTOSIS
AND PROSTATE
proteins except P21 increased (2-10-fold) in expression levels upon
serum starvation (Figs. 11 and 5D). Also like in NHP cells, the P21
levels decreased in a time-dependent manner, which was barely de
tectable by day 7 (Fig. 11). The only significant difference is that the
up-regulation of Bcl-XL in LNCaP cells was not as dramatic as in
NHP cells. Interestingly, the anti-P53 detected a lower band in addi
tion to the expected M, 53,000 protein. The molecular nature of this
lower band is unclear, although it might be a proteolytic product (48).
Survival of PC3 Cells in the Absence of Trophic Factors. PC3
cells behaved like PCA cells when deprived of serum growth factors.
These cells demonstrated a steady growth, which peaked on day 3
(Fig. \2A). The cell proliferation plateaued on day 4 (Fig. 1X4), when
increased apoptosis (DAPI labeling and DNA fragmentation) was
observed (Fig. 12, B and C, middle panel). This increased cell death
clearly negated the cell proliferation; therefore, the net number of live
cells did not further increase on day 4 (Fig. \2A). Starting from day 5,
the number of live cells began to decrease (Fig. \2A), suggesting that,
by this time, cell death (Fig. 125) began to surpass the cell prolifer
ation. Direct quantitation of apoptosis demonstrated that by day 8,
—¿60%
of the cells were apoptotic (Fig. 12, B and C, righi panel). This
loo-
20 •¿
0
1
2
3
4
5
CANCER CELL SURVIVAL
C
Time (days post growth factor withdrawal)
B
observation was consistent with the MTS measurement showing that
approximately the same number of cells (i.e., 100% cell number) as
initially input (i.e., 10,000 cells/well) were still alive (Fig. 12A) by day
8, considering that PC3 cells initially had a >2-fold proliferation (Fig.
12A). Western blotting examination of apoptosis proteins also re
vealed similar alterations as observed in PCA cells in that PC3 cells
i
Time (days post growth factor withdrawal)
Days after growth
factor withdrawal
0
1
Actin -/
P21
WAF-l
Bcl-2
Fig. 3. Survival of NHP cells in the absence of trophic factors. A. quantitation of live
cells using the MTS assays. The percentage cell number (compared to DO, which is 100%)
represents means derived from three independent experiments; bars, SE. B. quantitation
of apoptosis by DAPI staining. Inset, DNA fragmentation. C, micrographs showing
DAPI-stained NHP cells starved for 0 (¡eft).2 (middle), or 7 (right) days. Apoptotic cells
were brightly stained due to chromatin condensation. Note that in the middle and righi
panels, some apoptotic cells turned necrotic in culture and thus degraded. Therefore, an
overall decreased cell number was noticed. The arrowheads in the left and middle panels
illustrate apoptotic NHP cells, whereas the arrowheads in the right panel indicate very
few live cells. X100.
die immediately (as did NHP and BPH cells) until days 3-4 (Fig.
10ß).Prominent apoptosis (DAPI labeling and DNA fragmentation)
was observed around day 4 (Fig. 10, B and C, middle panel), yet the
total cell number (i.e., the same number as initially input) was main
tained up to day 6, after which the cell number went down (Fig. 10A).
These observations suggest that the cell loss due to apoptosis between
days 4-6 was partially compensated by cell proliferation. By day 8,
—¿70%
cells were apoptotic (Fig. 10, A-C, right panel). Immunoblotting examination of various apoptosis proteins revealed quite similar
patterns of alterations as observed in NHP cells. Specifically, all
Bax
Bak
—¿Â«.'
(22 kd)
Fig. 4. Alterations in apoptosis proteins in NHP cells after growth factor withdrawal.
Thirty u.g of whole-cell lysates prepared from unstarved NHP cells or from cells cultured
in the absence of growth factors for various days were resolved on a 12% SDS-PAGE. The
same membrane was sequentially probed with individual or mixed primary antibodies (see
Fig. 1). Left, individual molecules. The arrowhead on the right points to the M, —¿30.000
Bcl-XL.
3470
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APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
10.0
Vi
4)
a
a.
xv
w
'•O
£
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06
5.0
v
äs 2.5 0.0
DU
Dl
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D4
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D6
D7
DO
Dl
D2
Time (days)
DS
D6
e
^o
'Z
vt
o
o.
X
4)
D4
Time (days)
4-
W
D3
3-
s.
X
4)
4)
•¿^
2-
—¿
—¿
"w
i»
1-
—¿l—
DO
Dl
—¿l—
D2
D3
—¿l—
D4
—¿l—
DS
D6
DO
Dl
D2
D3
Time (days)
D4
DS
D6
D7
D8
Time (days)
o
VI
P21
Bcl-2
Bcl-XL
4-
Bak
Bad
vt
2o.
4l
hl
=X
9)
X
v
3-
W
2: 2H
—¿
"«3
W
ai
1-
DO
Dl
D2
D3
D4
DS
D6
D7
DO
Time (days)
Dl
D2
D3
Time
D4
DS
D6
D7
(days)
Fig. 5. Quantitative presentation of scanning data as determined by densitometry. The intensity of each individual protein band was first determined by scanning (41) and then
normalized to the intensity of actin protein band. After normalization, the individual protein levels are plotted as "relative expression" {i.e., fold changes) compared with unstarved cells
(i.e.. DO samples), the protein expression levels of which are considered as \.A, NHP cells. B. BPH cells. The values for P53 are: 1, 1.8, 9.7, 30.9, 15.4, 17.5, and 15 from DO to D6,
respectively. C, PCA cells. Note that the data for P53 were not plotted because the P53 protein on immunoblot overlapped with the P54/55 doublet. D, LNCaP cells. E. PC3 cells. The
values for Bcl-XL are: 1, 1.1, 0.9, 1.0, 1.0, 1.2, and 1.1 from DO to D7. respectively. F. Dul45 cells. Bcl-2 represents the M, 30,000 Bcl-2 protein band.
3471
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APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
with the M, 26,000 Bcl-2 protein (Fig. 13). In contrast, the Bcl-XL
protein did not change significantly (Figs. 13 and 5£).
Survival of Dul45 Cells in the Absence of Trophic Factors.
Dul45 cells demonstrated the highest survival potential (Fig. 144).
Upon serum removal, these cells gradually proliferated, nearly dou
bling the cell number by day 3 when DNA fragmentation was first
observed (Fig. 14ß),suggesting that the cell proliferation exceeded
cell death by apoptosis. The death rate in starved Du 145 cells dem
onstrated a very slow kinetic (Fig. 14ß),from ~10% on day 3, to
-15% on day 6 (Fig. 14C, middle), and to -25% by day 12, at which
time still about the same number (i.e., 10,000 cells/well initially
plated) of cells were alive (Fig. 14C, right panel). A portion (—5%)
1234567
Time (days post growth factor withdrawal)
of the Du 145 cells in the culture survived up to 30 days after serum
withdrawal (data not shown). Western blotting of apoptosis proteins
revealed that the proapoptotic proteins Bak and Bad did not undergo
significant changes in their expression levels (Figs. 15 and 5F), as in
Days after serum withdrawal
n
3
l
4
6
P53
234567
Time (days post growth factor withdrawal)
WAF-l
P21
Bcl-2
Bax
Fig. 6. Survival of BPH cells in the absence of trophic factors. A, quantitation of live
cells using the MTS assays. The percentage cell number (compared to DO. which is 100%)
represents means derived from three independent experiments; bars, SE. B, quantitation
of apoptosis by DAPI staining. Inset. DNA fragmentation. C micrographs showing
DAPI-stained NHP cells starved for 0 (left), 2 (middle), or 7 (right) days. Apoptotic cells
were brightly stained because of chromatin condensation. Note that in the middle and right
panels, some apoptotic cells turned necrotic in culture and thus degraded. Therefore, an
overall decreased cell number was noticed. The arrowheads in the left and middle panels
illustrate apoptotic BPH cells, whereas the arrowheads in the right panel indicate very few
live cells. X100.
lost their response in up-regulating all three proapoptotic proteins
upon serum deprivation (Figs. 13 and 5£).In fact, the Bax protein
actually went down dramatically by day 6 (Figs. 13 and 5£).Also
similar to PCA cells, the P21 levels significantly increased by day 3
prior to the initiation of DNA fragmentation (Figs. 13 and 5£).
Another aspect of the similarity between PC3 cells and PCA cells was
revealed by the detection of the P54/55 doublet in PC3 cells on day 5
after serum starvation (Fig. 13). Interestingly, the P25 band detected
in PCA cells was not observed in PC3 cells (Fig. 13). Different from
PCA cells, PC3 cells expressed the Mr 26,000 Bcl-2 protein, the levels
of which significantly increased upon serum starvation (Figs. 13 and
5E). The M, 27,000 Bcl-2 protein levels also elevated simultaneously
Bak
-
„¿
Bad
Actin
Fig. 7. Alterations in apoptosis proteins in BPH cells following growth factor with
drawal. Thirty ¿tgof whole cell lysates prepared from unstarved BPH cells or from cells
cultured in the absence of growth factors for various days were resolved on a 12%
SDS-PAGE. The same membrane was sequentially probed with individual primary
antibodies (see Fig. 1). Left, individual molecules. The arrowheads on the right point to
the M, -30,000 Bcl-X, and M, -22,000 Bad, respectively. Note the upper band in the
Bad blot was incompletely deprobed Bak.
3472
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APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
A 3fl0
(37). All three types of cells were diploid in karyotype, positive for
both keratinocytes and PSA, and negative for ras and p53 mutations
(37). When cultured in defined medium containing various growth
factors, these cells demonstrated progressively accelerating growth
properties; the population doubling times of NHP, BPH, and PCA
cells were 51, 37, and 29 h, respectively (37). Here we compared the
growth and survival properties of these primary prostate cells with
those of three established prostate cancer cell lines in the absence of
trophic factors.
We first characterized the expression of several apoptosis-related
proteins in different types of prostate cells, from normal to malignant.
All cells express P21, Bcl-XL, Bad, and Bak, and all cells except
Du 145 express Bax protein. However, primary cells generally express
little or no Bcl-2, whereas LNCaP cells express abundant Bcl-2
protein. In PC3 and Du 145 cells, a more complicated pattern of Bcl-2
expression was observed. In addition to the regular Mr 26,000 Bcl-2,
PC3 cells also express two minor upper bands that migrate at
M, 27,000 and M, 30,000, respectively. In contrast, Du 145 cells
25*'
200
lso
,„
50
2
4
6
g
10
12
Time (days post growth factor withdrawal)
Days post growth
factor withdrawal
23456789
1
0123451
i ii
6
Time (days post growth factor withdrawal)
P53
Actin -/
P21
P54/55
i
WAF-l
Bcl-2
Fig. 8. Survival of PCA cells in the absence of trophic factors. A, quantitation of live
cells using the MTS assays. The percentage cell number (compared to DO. which is 100%)
represents means derived from three independent experiments; bars, SE. B, quantitation
of apoptosis by DAP] staining. Inset, DNA fragmentation. C. DAPI-stained NHP cells
starved for 0 (left), 5 (middle), or 11 (right) days. Apoptotic cells were brightly stained due
to chromatin condensation. The arrowheads illustrate apoptotic PCA cells. X100.
Bcl-XL
PCA and PC3 cells. Also different from the PCA/PC3 cells, Du 145
did not express Bax protein, as tested by several antibodies raised
against different regions of the molecule (Fig. 15; data not shown).
The most salient feature is the expression of the Mr 30,000 Bcl-2
protein in Du 145 cells, the levels of which increased steadily after
serum starvation (Figs. 15 and 5F). Other aspects in the changes of
apoptosis proteins after serum starvation in Du 145 cells were similar
to those observed in PC3 cells. Specifically, P21 protein levels went
up (nearly 2-fold), whereas the Bcl-XL underwent a slight increase
(Figs. 15 and 5F). Also, like in PC3 cells, the P54/55 doublet was
detected in Du 145 cells on day 3 after serum starvation (Fig. 15).
Interestingly, the P25 protein band was also detected (Fig. 15).
DISCUSSION
Recently, NHP, BPH, and PCA cells were established, and their
growth properties and nutritional requirements were characterized
—¿â€”^
Bax
Bak
Bad
___.
Fig. 9. Alterations in apoptosis proteins in PCA cells after growth factor withdrawal.
Thirty fig of whole-cell lysates were prepared from unslarved PCA cells or from cells
cultured in the absence of growth factors for various days were resolved on a 12%
SDS-PAGE. The same membrane was sequentially probed with individual primary
antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to
the M, -30,000 Bcl-X,. Right, novel P25 and P54/55 proteins.
3473
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APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
treatment established the Mr 27,000 band but not the Mr 30,000 band
as the phosphorylated Bcl-2 protein. Therefore, the Mr 30,000 Bcl-2
protein identified in PC3 and Du 145 cells is different from the
Mr 30,000 Bcl-2 in HT29 cells, which is a hyperphosphorylated form
of the Mr 26,000 Bcl-2 (46). It is not yet known whether the Mr 30,000
Bcl-2 protein in PC3/Dul45 cells is or is related to the newly reported
Mr 30,000 BRAG-1, a Bcl-2-related protein primarily expressed in the
brain (47). Both Mr 27,000 and Mr 30,000 Bcl-2 proteins are detected
in PC3 and/or Du 145 cells without any prior stimulation, suggesting
that these proteins are endogenously expressed in both cells.
2468
Time (days post serum withdrawal)
B
10°
Days after serum withdrawal
I
01234567
MO 1 2 3 4 5 (days)
I
8
CO
4»
20
2468
P21
10
WAF-l
Time (days post serum withdrawal)
Bcl-2
Bel-XL
Bax
Fig. 10. Survival of LNCaP cells in the absence of trophic factors. A, quantitation of
live cells using the MTS assays. The percentage cell number (compared to DO, which is
100%) represents means derived from three independent experiments; bars, SE. B,
quantitation of apoptosis by DAPI staining. Inset, DNA fragmentation. C, DAPI-stained
NHP cells starved for 0 (left), 4 (middle), or 8 (right) days. Apoptotic cells were brightly
stained because of chromatin condensation. The arrowheads illustrate apoptotic LNCaP
cells. XI00.
^„.»-»-^^
Bak
Bad
appear to primarily express the Mr 30,000 band with very low levels
of the Mr 26,000 Bcl-2 protein. It is well known that Bcl-2 can
undergo phosphorylation on serine residues, resulting in either
Mr 27,000 Bcl-2 in multiple cell types such as leukemia, MCF-7, and
PC3 cells (42-46) or Mr 30,000 Bcl-2 in HT-29 colon cancer cells
(46). The Mr 30,000 Bcl-2 protein has not been reported in either PC3
or Du145 cells, even after treatment with taxol or okadaic acid (42,43,
45). We therefore further characterized the Mr 27,000 and A/r 30,000
bands detected in PC3 and/or Du 145 cells by the monoclonal antiBcl-2 antibody. Immunoprecipitation followed by Western blotting
first established the Mr 30,000 band in both cell types as a specific
Bcl-2-related protein instead of nonspecific antibody binding. Subse
quently, a dephosphorylation experiment with alkaline phosphatase
Fig. 11. Alterations in apoptosis proteins in LNCaP cells after growth factor with
drawal. Thirty (ig of whole-cell lysates prepared from unstarved LNCaP cells or from
cells cultured in the absence of serum for various days were resolved on a 12%
SDS-PAGE. The same membrane was sequentially probed with individual primary
antibodies (see Fig. 1). Left, individual molecules. The arrowhead on the right points to
the Mr -30,000 Bcl-XL. Note that the anti-P53 detected the expected M, 53,000 protein
and a lower band, whose identity is presently unknown.
3474
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APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
in the absence of trophic factors, than in PC3/PCA/Dul45 cells
(summarized in Fig. 16), suggesting that cancerous prostate cells
naturally possess a longer life span (or survivability) than NHP/BPH
cells.
LNCaP cells, initially isolated from the lymph node metastasis of a
prostate cancer patient, demonstrate a survivability between that of
NHP/BPH and that of PC3/PCA/Dul45 cells. These cells behave
differently from other cancer cells in that they do not proliferate
significantly in the absence of trophic factors. However, LNCaP cells
also behave differently from NHP/BPH cells in that they do not die
immediately after serum withdrawal. Instead, these cells keep a rela
tively constant cell number for 5-6 days, although the apoptosis is
initiated at approximately day 4. The mechanism(s) for the unique
survival characteristics of LNCaP cells are presently unknown. Taken
together, the present data suggest that prostate cancer cells generally
demonstrate longer survivability than their normal counterparts due to
increased proliferative capability and decreased cell death rate. The
increased cell proliferation results from the production by cancer cells
of various growth factors (49), whereas the decreased death rate
300
250
200
150
100 *
2
4
6
H
Time (days after serum withdrawal)
B
100
80
L
co
ä.
•¿<
20"
.2
Days post serum
withdrawal
6543210
Days after serum withdrawal
2468
10
6
234
7
Time (days after serum withdrawal)
P21
WAF-l
_._ _ «M
* =:
P54/55
©-Bcl-2 (27 kd)
Bcl-2
Bcl-Xt
Bax
Fig. 12. Survival of PC3 cells in the absence of trophic factors. A. quantitation of live
cells using the MTS assays. The percentage cell number (compared to DO, which is 100%)
represents means derived from six independent experiments; bars, SE. B. quantitation of
apoptosis by DAP1 staining. Inset. DNA fragmentation. C, DAPl-stained NHP cells
starved for 0 (left). 4 (middle), or 8 (right) days. Apoptotic cells were brightly stained due
to chromatin condensation. X 100.
Growth and Survival of Various Prostate Cells upon Trophic
Factor Withdrawal. NHP and BPH cells do not grow and demon
strate the lowest survival capacity upon trophic factor withdrawal.
Apoptosis is observed in these cells as early as 24 h after growth
factor withdrawal. In contrast, primary (PCA) and metastatic (PC3
and Dul45) carcinoma cells survive much longer, and these cells keep
dividing and proliferating in the absence of trophic factors. These
three cancer cell types follow very similar growth/survival patterns
upon removal of trophic factors: an initial aggressive growth phase
(the first 2-3 days) in which apoptosis rarely occurs; a second "coun
terbalancing" phase in which cell loss by apoptosis (taking place on
days 3-4) is compensated by sustained cell proliferation; and a third
"death" phase in which apoptosis surpasses the cell growth. Direct
measurement of cell death by DAPI staining and MTT assays (not
shown) has demonstrated a much higher death rate in NHP/BHP cells.
Bak
—¿
Bad
Actin
_ Ä
_.
. ^
Fig. 13. Alterations in apoptosis proteins in PC3 cells after scrum withdrawal. Thirty
Hg of whole-cell lysates prepared from unstarved PC3 cells or from cells cultured in the
absence of serum for various days were resolved on a 12% SDS-PAGE. The same
membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left,
individual molecules. The arrowhead on the right points to the M, -30.000 Bcl-X,. The
Bcl-2 proteins and Ihe P54/55 doublet are also labeled on the riditi. Note that Lanes 2 and
3 (i.e., days I and 2) loaded less protein, as evidenced by the actin band. Thus, the
intensities of these two lanes and all of the rest of the lanes were normali/ed against that
of the actin band.
3475
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APOPTOSiS
AND PROSTATE
trum of human tumor cells (4). In this study, there appears to have a
correlation between the P53 response and apoptotic sensitivity of
various prostate cells. NHP, BPH, and LNCaP cells, which are the cell
types sensitive to apoptosis induction by trophic factor withdrawal,
demonstrated a time-dependent increase in P53 protein expression.
BPH cells demonstrated the most robust (~30-fold increase by day 3)
and longest up-regulation of P53, and these cells are most sensitive to
apoptosis induction. In contrast, PCA cells, although expressing wildtype P53, do not demonstrate up-regulation of P53, and these cells
survive much longer than NHP, BPH, and LNCaP cells (Fig. 16).
Similarly, PC3 and Dul45 cells, which do not express functional P53,
also possess longer survivability in the absence of trophic factors.
Taken together, these observations correlate an up-regulation of func
tional P53 with increased sensitivity of prostate cells to apoptosis
induction by deprivation (Fig. 16).
Quite contrasting alterations are also seen with P21 in sensitive
3
2468
10
Time (days after serum withdrawal)
B
100
CANCER CELL SURVIVAL
Days post serum withdrawal
0123-156
Ml
Days after serum withdrawal
40
012345
2468
P21
10
67
-
WAF-l
Time (days after serum withdrawal)
P54/55
Bcl-2
30kd-Bcl-2
Bel-XL
Bax
Fig. 14. Survival of Du 145 cells in the absence of trophic factors. A. quantitation of live
cells using the MTS assays. The percentage cell number (compared to DO. which is 100%)
represents means derived from four independent experiments: bars, SE. B, quantitation of
apoptosis by DAPI staining. Insel, DNA fragmentation. C, DAPI-stained NHP cells
starved for 0 (left}, 6 (middle), or 12 (righi) days. Apoptotic cells were brightly stained
because of chromatin condensation. X100.
results from evasion of apoptosis. It is of note that PCA, one of the
primary cell types used, demonstrates even higher survivability than
LNCaP and PC3 cells, which are established cell lines kept in culture
for many generations. Therefore, it is unlikely that any of the differ
ences observed among these cells may have resulted from tissue
culture artifacts.
Role of p53 and P21 in Prostate Cell Growth/Apoptosis
upon
Trophic Factor Withdrawal.
p53 plays two essential roles in main
taining the cellular homeostasis: introducing "checkpoints" to arrest
cell cycle; and triggering apoptosis to self-destroy those cells beyond
repair (50). p53 mediates its biological functions by transcriptionally
regulating (either activating or inhibiting) many of its targets, among
which is p21, a CIP/KIP family cyclin-dependent kinase inhibitor. In
response to genotoxic shocks, cells generally halt cell cycle progres
sion by up-regulating P21 protein in a p53-dependent manner. Overexpression of wild-type p53 induces apoptotic death of a wide spec-
Bad
Actin
—¿
•¿â€¢Â»
—¿
—¿
—¿â€¢
«
Fig. 15. Alterations in apoptosis proteins in Du 145 cells after serum withdrawal. Thirty
fig of whole-cell lysates prepared from unstarved Du 145 cells or from cells cultured in the
absence of serum for various days were resolved on a 12% SDS-PAGE. The same
membrane was sequentially probed with individual primary antibodies (see Fig. 1). Left,
individual molecules. The arrowhead on the right points to the A/r —¿30,000
Bcl-X,. The
M, 30,000 Bcl-2, P25, and P54/55 doublet are also labeled on the right. Note that the
regular Mr 26.000 Bcl-2 protein was barely detectable in this experiment. Note that Lane
2 (i.e., day 1) loaded less protein, as evidenced by actin band. Thus, the intensities of this
lane and all of the rest of the lanes were normalized against that of the actin band.
3476
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APOPTOSIS AND PROSTATE CANCER CELL SURVIVAL
mutantP21:A
—¿
ABcl-XL:
Bcl-2:
Bcl-2:
ABcl-XL:
p53:iP21:-Bcl-2: p53:AP21:fBcl-2:
RiiBcl-2:
ABad/Bak:
little/noBcl-XLlBax/Bad/Bak:
—¿Bad/Bak:
fBcl-2:
1:
—¿Bax:
little/noBcl-XL: A,Bcl-XL:ABax/Bad/Bak:
little/noBcl-XL:ABax/Bad/Bak:AGrowth
—¿Bax:JP25/P54/55:
—¿P25/P54/55: deletedP25/P54/55:
A.Bax/Bad:
yesGrowth
yesGrowth
yesGrowth
AGrowth
AGrowth
+++Apop
-Apop
-Apop
+++Apop
+++Apop
+Apop
+•Dul45
++£PC3~^0wtp53:
+•
+++BPH
+++NHP
++i
nullP21:A
Awt
Fig. 16. A, the overall survivability of various
prostate cells in the absence of trophic factors is
presented: BPH < NHP < LNCaP < PC3 < PCA
< Dul45. Shown above are changes in apoptosis
proteins. Refer to text for details. —¿,
no change;
upward arrowhead, increase; downward arrow
head, decrease; little/no, little or no expression.
Shown in the middle are the cell growth and apop
tosis properties of various prostate cells in the ab
sence of trophic factors, as deduced from preceding
figures. —¿,
no growth. + to +4-+, different levels
of apoptosis. BPH and NHP cells do not proliferate
at all and demonstrated fast apoptotic kinetics. LNCaP cells demonstrated a low level of proliferation
and an intermediate level of apoptosis. PC3 cells
demonstrated very aggressive growth and interme
diate levels of apoptosis. PCA and Du 145 cells, in
contrast, demonstrated a rapid cell proliferation as
well as a much delayed and decreased levels of
apoptosis. B, summary of molecular events leading
to differential survivabilities of normal versus can
cerous prostate epithelial cells in the absence of
trophic factors. Note thai there exist exceptions to
this generalized scheme. For example, PCA cells
possess wild-type P53 and express little/no Bcl-2.
-^wt
B
~^0wtp53:AP2
LNCaP-^p53:
PCA -^p53:
Normal Prostate Epithelium
androgen-dependence
growth factor dependence
long
timewild-type
population doubling
upregulation
P53
P21: no change or decrease
litue/no Bcl-2DeprivationDeprivationP53
Bax/Bad/Bakupregulalion
Bcl-2: little/no expression
Prostate
No cell
proliferation
•¿Apoptosis
>V Decreased
.—^ cell number
Cancer Cells
androgen-independence
£e^No
autocrine growth factor production
short population doubling
timenon-functional
P53 upregulalion
—¿
^ survivabili ty
P53
of -^
P21 upregulation
Bcl-2low/no
high
BaxDeprivationDeprivation Bax/Bad/Bak:decrease .^
apoptosisT^Extended
or deletion
Bcl-2: upregulation
Novel proteins: P25;
P54/55 (?)proliferationEvasion
calcineurin), or the death-promoting
activity of Bad (through
Raf-1; Refs. 55 and 56). The precise mechanism(s) for the antideath effects of Bcl-2/Bcl-XL is still unknown and most likely is
cell type and inducer dependent. Bcl-2 has been shown to be
phosphorylated by Raf-1 and some other protein kinases, and this
phosphorylation has been hypothesized to inactivate the cytoprotective effect of Bcl-2 (42-46). In addition, both Bcl-2/Bcl-XL and
Bax have been shown to be transcriptionally regulated by p53 (57,
58). Increased Bcl-2/Bcl-X, protein levels are generally associated
and P21 in apoptosis of prostate cells is independent of each other, as
with cytoprotection
and extended cell survival. In this study,
reported by others in oncogene-mediated fibroblast apoptosis (52);
NHP/BPH/PCA cells express little or no Bcl-2, and growth factor
withdrawal fails to up-regulate Bcl-2, although all three cell types
and (c) increased P21 expression is protecting PCA/PC3/Dul45 cells
express wild-type p53. In contrast, Bcl-2 in LNCaP (wild-type
from apoptosis, as observed in other cell systems (53, 54). The fact
that P21 levels go up before apoptosis suggests a causal rather than a p53) and PC3 cells (p53-null) steadily and significantly increases
coincidental relationship between the two phenomena. The increased
upon deprivation. More interestingly. Du 145 cells primarily ex
press the Mr 30,000 Bcl-2. a very stable protein, the levels of which
P21 could be inducing a cell cycle arrest because the peak level of
induction of this protein also coincides with the time when cells stop
remain persistently elevated throughout the starvation period. With
respect to Bcl-X,, all cells except PC3 demonstrate a certain
proliferation. It is known that dividing cells are much more prone to
degree
of up-regulation of this protein in response to starvation.
apoptosis induction (55); therefore, it is quite likely that the increased
P21 will put a "brake" on the proliferative machinery in prostate
Based on these results, it appears that; (a) the up-regulation of
cancer cells to avoid "mitotic catastrophe" (55).
Bcl-2 and/or Bcl-X, in various prostate cells represents a general
Role of Bcl-2 and Bcl-X, in Prostate Cell Growth/Apoptosis
defensive response to stresses such as deprivation. The up-regula
upon Trophic Factor Withdrawal.
Bcl-2 and Bcl-XL are two
tion of Bcl-X, may be the major defensive (survival) mechanism in
members in the Bcl-2 family that possess anti-apoptosis functions.
NHP cells; the significant up-regulation of this protein in NHP
Many biological activities have been assigned to these proteins for
cells may also explain why these cells are relatively more resistant
their death-sparing effects: forming membrane channels; modulat
to apoptosis induction than BPH cells. However, the cytoprotective
ing mitochondria permeability transition pore; retarding cytoeffect of up-regulated Bcl-XL will be overridden by the simulta
chrome c and/or AIF apoptosis-inducing
factor release from mito
neous up-regulation of Bax, Bad. and Bak: (/?) the up-regulation of
Bcl-2 and/or Bcl-X, may not depend on p53: and (c) the differ
chondria; working as antioxidants; preventing calcium fluctuation;
ential up-regulation of Bcl-2 and/or Bcl-X, in various prostate
sequestering proapoptotic proteins (such as Bax. Bak, and Bik);
and indirectly modulating caspase activity (through CED-4). cell
cells might partially contribute to their differential survival abili
cycle (through p53BP-2), NF-AT transcriptional activity (through
ties (Fig. 16).
3477
(i.e.. BPH, NHP, and LNCaP) versus resistant (i.e.. PCA, PC3, and
Du 145) cells. In the former, upon trophic factor withdrawal, P21
either does not change significantly (BPH) or gradually decreases
(NHP and LNCaP) while in the latter; the P21 levels invariably
increase either prior to or simultaneously with the appearance of DNA
fragmentation. These quite distinct changes in the two groups of cells
suggest that: (a) the P21 expression is unrelated to or independent of
the P53 status, which is consistent with data showing that P21 ex
pression can be p53-independent (50, 51); (b) the involvement of P53
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research.
APOPTOSIS
AND PROSTATE
CANCER CELL SURVIVAL
Role of Bax, Bad, and Bak in Prostate Cell Growth/Apoptosis
upon Trophic Factor Withdrawal. In contrast to Bcl-2 and Bcl-XL,
proapoptotic proteins Bax, Bad, and Bak demonstrate very distinct
alterations in their expression levels that highly correlate with the
sensitivity to apoptosis induction in individual cell types (Fig. 16).
Specifically, upon growth factor withdrawal, all sensitive cells, i.e.,
NHP, BPH, and LNCaP, demonstrate a time-dependent up-regulation
of these proteins. In contrast, long-living cells (PCA, PC3, and
Du 145) invariably demonstrate a loss of this up-regulation response.
More dramatically, PC3 cells demonstrate a gradual decrease, and
Du 145, the most resistant cells, demonstrate a complete loss in Bax
expression. The Bax/Bad/Bak protein expression appears to be cor
related with the p53 functionality; their levels are increased in cells
with wild-type p53 (NHP, BPH, and LNCaP), and their levels show
no increase or show decrease or loss in cells with no or mutant p53
(PC3 and Du 145). PCA cells, although expressing functional p53,
somehow do not up-regulate this protein in response to deprivation
and therefore resemble PC3 and Du 145 cells in terms of p53 func
tionality.
Role of Novel Proteins in Prostate Cell Growth/Apoptosis upon
Trophic Factor Withdrawal. In addition to the novel M, 30,000
Bcl-2 protein detected in Du 145 cells, several other proteins, specif
REFERENCES
i. Raff. M. C. Social controls on cell survival and cell death. Nature (Lond.), 356:
397-400, 1992.
Raff. M. C.. Barres. B. A., Bume, J. F., Coles, H. S.. Ishizaki, Y., and Jacobson, M. D.
Programmed cell death and the control of cell survival: lessons from the nervous
system. Science (Washington DC), 262: 695-700. 1993.
Denmead. S. R., Lin, X. S.. and Isaacs. J. T. Role of programmed (apoptotic) cell
death during the progression and therapy for prostate cancer. Prostate, 28: 251-256.
1996.
Tang. D. G., and Porter, A. T. Target to apoptosis: a hopeful weapon for prostate
cancer. Prostate, 32: 284-293, 1997.
McDonnell, T. J.. Troncoso, P., Brisby, S. M., Logothetis, C. L., Chung, L. W. K.,
Hsieh, J. T., Tu. S. M., and Campbell, M. L. Expression of the protooncogene Bcl-2
in thèprostate and its association with emergence of androgen-independent prostate
cancer. Cancer Res., 52: 6940-6944, 1992.
Colombe!, M., Symmans, F., Gil, S., O'Toole, K. M., Choplin, D., Benson, M.,
Olsson. C. A.. Korsmeyer. S., and Buttyan. R. Detection of the apoptosis-suppressing
oncoprotein Bcl-2 in hormone-refractory human prostate cancers. Am. J. Pathol.. 143:
390-400, 1993.
Liu, A. Y., Corey, E., Bladou, F., Lange, P. H., and Vessella, R. L. Prostatic cell
lineage markers: emergence of Bcl2* cells of human prostate cancer xenograft
10.
ically P25 and P54/55 doublet, are also detected in PCA, PC3, and
Du 145 cells. These proteins appear together at the same time and can
be detected by at least two unrelated monoclonal antibodies, i.e.,
monoclonal anti-P53 (in PCA cells; Fig. 9) and monoclonal anti-Bcl-2
(in PC3 and Du 145 cells; Figs. 13 and 15). Based on these charac
teristics and their apparent molecular weights, these proteins appear to
be immunoglobulin heavy (M, -55,000) and light (M, -25,000)
chains (see also Fig. 2). Why these "immunoglobulin molecules" are
up-regulated is unclear. However, the fact that they are only upregulated in resistant cells (PCA, PC3, and Dul45) and their regula
tion follows a distinct pattern (i.e., around the time when cells initiate
apoptotic programs) point to an intriguing possibility that they might
be causally involved in extending the survival of prostate cancer cells.
Synopsis. The present study uses a simple model to compare the
growth/survival characteristics of normal versus cancerous prostate
cells cultured in the absence of trophic factors. Fig. 16 summarizes the
sequential molecular events that occur as these cells progress from a
sensitive to a resistant stage with respect to apoptosis induction. NHP
and BPH cells express wild-type p53 and little/no Bcl-2. In response
to growth factor withdrawal, these cells stop proliferation and rapidly
up-regulate p53, which may then up-regulate proapoptotic proteins
Bax/Bad/Bak. Cells rapidly enter an apoptotic phase. In contrast,
prostate cancer cells, upon deprivation, still proliferate aggressively
due to autocrine production of various growth factors. These cells
(except PCA) endogenously express much higher levels of Bcl-2.
They express either mutant p53 (PC3 and Dul45) or wild-type p53,
which somehow does not respond to starvation by up-regulating its
expression (PCA), leading to the loss of up-regulation or decrease of
proapoptotic proteins. Furthermore, cancer cells such as Du 145 even
completely delete a proapoptotic protein (i.e., Bax). At the same time,
these cells up-regulate P21, which could induce cell cycle arrest at an
appropriate time (because proliferating cells are generally more sen
sitive to apoptosis induction; Ref. 55), and Bcl-2, which by itself
could extend cell survival. Last but not least, the long-living cells
express some novel immunoglobulin or immunoglobulin-like proteins
that might also help maintain the cell survivability. Cells with inter
mediate life span, i.e., LNCaP, demonstrate a variety of characteristics
lying between the above two cell types. Sustained proliferation, to
gether with multiple molecular mechanisms to evade apoptosis, leads
to significantly extended survivability of prostate cancer cells, com
pared with their normal counterparts (Fig. 16).
12.
14.
15.
17.
18.
19.
20.
22.
23.
24.
25.
LuCaP 23 following castration. Int. J. Cancer, 65: 85-89. 1996.
Fuyuya, Y., Krajewski, S., Epstein. J. !.. Reed, J. C., and Isaacs. J. T. Expression of
Bcl-2 and progression of human and rodent prostatic cancers. Clin. Cancer Res.. 2:
389-398, 1996.
McConkey, D. J.. Greene. G.. and Pettaway. C. A. Apoptosis resistance increases with
metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer
Res., 56: 5594-5599, 1996.
Berchem, G. J., Bossier. M., Sugars, L. Y.. Voeller. H. J., Zeitlin. S.. and Gelman.
E. P. Androgens induce resistance to bcl-2-mediated apoptosis in LNCaP prostate
cancer cells. Cancer Res., 55: 735-738, 1995.
Raffo, A. J., Perlman, H.. Chen. M-W., Day, M. L., Stritman, J. S., and Buttyan. R.
Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and
confers resistance to androgen depletion in vivo. Cancer Res., 55: 4438-4445, 1995.
Isaacs. W. B., Carter. B. S.. and Ewing, C. M. Wild-type p53 suppresses growth of
human prostate cancer cells containing mutant p53 alÃ-eles.Cancer Res.. 51: 47164720. 1991.
Eastman, J. A.. Hall, S. J.. Sehgal. Wang, I. J.. Tirarne, T. L., Yang, G.. ConnellCrowley, L.. Elledge. S. J., Zhang, W-W.. Harper. J-W.. and Thompson, T. C. In vivo
gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res.. 55:
5151-5155. 1995.
Yang. C., Cirielli, C., Capogrossi, M. C., and Passanti. A. Adenovirus-mediated
wild-type p53 expression induces apoptosis and suppresses tumorigenesis of prostatic
tumor cells. Cancer Res., 55: 4210-4213, 1995.
Sells, S. F.. Muthukkumar. S., Maddiwar, N.. Johnstone. R., Boghaert. E., Gillis. D.,
Liu, G.. Nair. P., Monnig. S., Collini. P.. Mattson, M. P., Sukhatme, V. P., Zimmer,
S. G., Wood, D. P., Jr., McRoberts, J. W., Shi, Y., and Rangnekar, V. M. Expression
and function of the leucine zipper protein par-4 in apoptosis. Mol. Cell. Biol., 17:
3823-3832. 1997.
Day, M. L., Foster, R. G.. Day. K. C., Zhao, X.. Humphrey, P., Zhang. S. H.. and
Dean, D. C. Cell anchorage regulates apoptosis through the retinoblastoma tumor
suppressor/E2F pathway. J. Biol. Chem., 272: 8125-8128. 1997.
Hsing. A. Y., Kadomatsu. K., Bonham. M. J.. and Daneilpour. D. Regulation of
apoptosis induced by transforming growth factor-ßl in nontumorigenic and tumorigenie rat prostatic epithelial cell lines. Cancer Res., 56: 5146-5149, 1996.
Sensibar, J. A., Sutkowski. D. M., Raffo, A.. Buttyan, R.. Griswold, M. D., Sylvester.
S. R., Kozlowski. J. M.. and Lee, C. Prevention of cell death induced by tumor
necrosis factor a in LNCaP cells by Overexpression of sulfated glycoprotein-2
(clusterin). Cancer Res.. 55: 2431-2437, 1995.
Rokhlin, O. W.. Bishop, G. A.. Hostager, B. S., Waldschmidt. T. J.. Sidorenko, S. P..
Pavloff. N., Kiefer, M. C., Umanski, S. R., Glover, R. A., and Cohen, M. B.
Fas-mediated apoptosis in human prostatic carcinoma cell lines. Cancer Res., 57:
1758-1768. 1997.
Borner. M. M., Myers, C. E., Sartor, O., Sei, Y„Toko, T., Trepel, J. B.. and
Schneider. E. Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line
PC-3. Cancer Res., 53: 2122-2128, 1995.
Sinha, B. K.. Yamazaki, H., Eliot, H., Schneider. E.. Borner, M. M.. and O'Connor,
P. M. Relationships between proto-oncogene expression and apoptosis induced by
anticancer drugs in human prostate tumor cells. Biochim. Biophys. Acta. 7270:
12-18. 1995.
Vukanovic, J., and Isaacs, J. T. Human prostatic cancer cells are sensitive to
programmed (apoptotic) death induced by the antiangiogenic agent Linomide. Cancer
Res., 55: 3517-3520, 1995.
Li, C. J., Wang, C., and Pardee, A. B. Induction of apoptosis by ß-lapachone in
human prostate cancer cells. Cancer Res., 55: 3712-3715, 1995.
Planchón, S. M., Wuerzberger, S.. Frydman. B., Witiak. D. T., Hutson, P.. Church,
D. P.. Wilding. G.. and Boothman, D. A. /3-Lapachone-mediated apoptosis in human
promyelocytic leukemia (HL-60) and human prostate cancer cells: a p53-independent
response. Cancer Res., 55: 3706-3711, 1995.
Carducci, M. A., Nelson. J. B.. Chan-Tack, K. M., Ayyagari, S. R.. Sweatt, W. H.,
Campbell. P. A.. Nelson. W. G., and Simons, J. W. Phenylbutyrate induces apoptosis
in human prostate cancer and is more potent than phenylacetate. Clin. Cancer Res., 2:
379-387. 1996.
3478
Downloaded from cancerres.aacrjournals.org on April 29, 2017. © 1998 American Association for Cancer Research.
APOPTOSIS
26. Wright. P. S.. Cross-Doersen.
AND PROSTATE
CANCER CELL SURVIVAL
D.. Th'ng. J. P. H., Guo, X-W., Crissman, H. A..
Bradbury. E. M., Montgomery. L. R.. Thompson, E. Y.. Loudy. D. E., Johnston. J. O..
and Bitoni. A. J. A ribonucleotide reducÃ-aseinhibitor. MDL 101,731, induces apoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts. Exp.
Cell Res., 222.- 54-60, 1996.
27. Robertson, C. N.. Robertson. K. M.. Padilla, G. M., O'Brien, E. T., Cook, J. M., Kim,
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
C-S., and Fine, R. L. Induction of apoplosis by dielhylstilbestrol in hormoneinsensitive prostate cancer cells. J. Nati. Cancer Inst., X8: 908-917. 1996.
Grove, K. L., and Cheng, Y-C. Uptake and metabolism of the new anticancer
compound ß-L-(
- )-dioxolane-cytidine in human prostate carcinoma DU-145 cells.
Cancer Res., 56: 4187-4191, 1996.
Robertson, K. M.. Penland. S. N., Padilla. G. M., Selvan, R. S.. Kim, C-S., Fine. R. L.,
and Robertson, C. N. Fenretinide. Induction of apoptosis and endogenous transform
ing growth factor ßin PC-3 prostate cancer cells. Cell Growth Differ.. 8: 101-111,
1997.
Day, M. L., Zhao, X.. Wu, S., Swanson, P. E.. and Humphrey, P. A. Phorbol
ester-induced apoptosis is accompanied by NGF1-A and c-fos activation in androgensensitive prostate cancer cells. Cell Growth Differ.. 5: 735-741. 1994.
Powell, C. T., Brittis. N. J.. Stec, D.. Hug. H.. Heston, W. D. W., and Fair, W. R.
Persistent membrane translocation of protein kinase Ca during 12-O-tetradecanoylphorbol-13-acetate-induced
apoptosis of LNCaP human prostate cancer cells. Cell
Growth Differ., 7: 419-428, 1996.
Rusnak, J. M., and Lazo. J. S. Downregulation of protein kinase C suppresses
induction of apoptosis in human prostatic carcinoma cells. Exp. Cell Res., 224:
189-199, 1996.
Huyuya. Y., Lundmo. P.. Short, A. D., Gill, D. L.. and Isaacs, J. T. The role of
calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgenindependent prostatic cancer cells induced by thapsigargin. Cancer Res., 54: 61676175, 1994.
Garde. S.. Basrur. V.. Finkelman. M.. Li. L.. Krishan. A.. Ben-Josef. E.. Haddad. M..
Taylor. J. D., Porter, A. T.. and Tang. D. G. Prostate secretory protein (PSP94)
suppresses the growth of human androgen-independent prostate adenocarcinoma cells
in vitro and in \'ivu by inducing apoptosis. Proc. Am. Soc. Clin. Oncol., 76; A1970,
1997.
Tang. D. G.. Li, L., Zhu, Z., and Joshi, B. Apoptosis in the absence of cytochrome r
accumulation in the cytosol. Biochem. Biophys. Res. Commun.. 242: 380-384, 1998.
Tang, D. G., Li. L., Zhu, Z.. Joshi. B.. Johnson, C. R., Marnett. L. J.. Honn. K. V.,
Crissman. J. D.. timar. J., and Porter. A. T. BMD188. a novel hydroxamic acid
compound, demonstrates potent anti-prostate cancer effects m vitro and in vivo:
requirements for mitochondria, reactive oxygen species, and proteases. Pathol. Oncol.
Res., in press, 1998.
Chopra, D. P., Grignon, D. J., Joiakim, A., Mathieu, P. A.. Mohamed, A., Sakr, W. A.,
Powell. I. J.. and Sarkar. F. H. Differential growth factor responses of epithelial cell
cultures derived from normal human prostate, benign prostatic hyperplasia and
primary prostate carcinoma. J. Cell Physiol., 169: 269-280, 1996.
Tang. D. G., Chen, Y. Q.. and Honn. K. V. Arachidonate lipoxygenases as essential
regulators of cell survival and apoptosis. Proc. Nati. Acad. Sci. USA. 93: 5241-5246,
1996.
Tang, D. G.. and Honn, K. V. Apoptosis of W256 carcinosarcoma cells of the
monocytoid origin induced by NDGA involves lipid peroxidation and depletion of
40.
41.
42.
43.
44.
45.
46.
GSH: role of 12-lipoxygenase in regulating tumor cell survival. J. Cell Physiol., 172:
155-170, 1997.
Mo.sman. T. Rapid colorimetrie assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods. 65: 55-63. 1983.
Tang. D. G., Chen, Y. Q.. Newman. P. J.. Shi, L., Gao, X.. Diglio. C. A., and Honn,
K. V. Identification of PECAM-1 in solid tumor cells and its potential involvement
in tumor cell adhesion to endothelium. J. Biol. Chem., 268: 22882-22894. 1993.
Haldar, S., Jena, N., and Croce, C. M. Inactivation of Bcl-2 by phosphorylation. Proc.
Nati. Acad. Sci. USA. 92: 4507-4511. 1995.
Haldar. S., Chintapalli. J.. and Croce. C. M. Taxol induces hcl-2 phosphorylation and
death of prostate cancer cells. Cancer Res., 56: 1253-1255, 1996.
Chen. C-Y.. and Faller. D. V. Phosphorylation of Bcl-2 protein and association with
p2lR" in Ras-induced apoptosis. J. Biol. Chem.. 271: 2376-2379, 1996.
Blagoskonny, M. V.. Schulte. T.. Nguyen, P.. Trepel. J.. and Neckers. L. M. Taxolinduced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and repre
sents a novel c-Raf-1 signal transduction pathway. Cancer Res.. 56: 1851-1854.
1996.
Guan. R-J.. Moss. S. F.. Arber. N.. Krajewski, S., Reed. J. C.. and Holt. P. R. 30 Kd
phosphorylated form of Bcl-2 protein in human colon. Oncogene. 12: 2605-2609,
1996.
47. Das. R.. Reddy. E. P., Chatterjee, D., and Andrews, D. W. Identification of a novel
Bcl-2 related gene, BRAG-1. in human glioma. Oncogene, 12: 947-951. 1996.
48. Kubbutat. M. H. G., and Vousden, K. H. Proteolytic cleavage of human p53 by
calpain: a potential regulator of protein stability. Mol. Cell. Biol., 17: 460-468. 1997.
49. Culic, Z.. Hobisch, A., Cronauer, M. V.. Radmayr, C., Hittmair, A., Zhang, J..
Thumher. M.. Bartsch. G.. and Klocker. H. Regulation of prostatic growth and
function by peptide growth factors. Prostate, 28: 392-405, 1996.
50. Ko. L. J.. and Prives. C. P53: puzzle and paradigm. Genes Dev.. 10: 1054-1072,
1996.
51. Levine, A. p53, the cellular gatekeeper for growth and division. Cell, 88: 323-331,
1997.
52. Attardi. L. D., Lowe. S. W.. Brugarolas. J., and Jacks. T. Transcriptional activation by
p53. but not induction of the p21 gene, is essential for oncogene-mediated apoptosis.
EMBO J.. 15: 3693-3701, 1996.
53. Gorospe, M.. Cirielli, C., Wang, X., Seth, P.. Capogrossi, M. C.. and Holbrook, N. J.
P21Waf-l/Cipl protects against p53-mediated apoptosis of human melanoma cells.
Oncogene. 14: 929-935. 1997.
54. Polyak. K., Waldman. T.. He. T-C., Kinzler, K. W., and Vogelstein, B. Genetic
determinants of p53-induced apoptosis and growth arrest. Genes Dev., 10: 19451952, 1996.
55. Reed, J. C. Double identity for proteins of the Bcl-2 family. Nature (Lond.), 387:
773-776, 1997.
56. Hockenbery, D. M. Bcl-2, a novel regulator of cell death. BioEssays, 17: 631-638,
1995.
57. Merchant, A. K.. Loney, T. L.. and Maybaum. J. Expression of wild-type p53
stimulates an increase in both Bax and Bcl-X, protein content in HT29 cells.
Oncogene. 13: 2631-2637. 1996.
58. Miyashita, T., Krajewski, S., Krajewski, M., Wang, H. G., Lin, H. K., Liebermann,
D. A.. Hoffman, B.. and Reed, J. C. Tumor suppressor p53 is a regulator of hcl-2 and
box gene expression in vitro and in vivo. Oncogene. 9: 1799-1805, 1994.
3479
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Extended Survivability of Prostate Cancer Cells in the Absence
of Trophic Factors: Increased Proliferation, Evasion of
Apoptosis, and the Role of Apoptosis Proteins
Dean G. Tang, Li Li, Dharam P. Chopra, et al.
Cancer Res 1998;58:3466-3479.
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